The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities

Tatsunori Maekawa Sayuri Mori Yui Sasaki Takashi Miyajima Sadahiro Azuma Etsuro Ohta Fumiya Obata 0 0 R & D Center for Cell Design, Institute for Regenerative Medicine and Cell Design, Kitasato University School of Allied Health Sciences, 2) Division of Immunology, Kitasato University School of Allied Health Sciences , Kitasato 1-15-1, Sagamihara Minami-ku, Kanagawa 252-0373 , Japan Background: Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson's disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family. Results: The TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylase-positive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls. Conclusions: The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation. - Background Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinsons disease (PD), PARK8, which originally has been defined by linkage analysis of a Japanese family (Sagamihara family) [1-4]. LRRK2 is a complex kinase consisting of LRR, ROC, COR, kinase, and WD40 domains [5]. The Sagamihara family patients have the I2020T mutation in the kinase domain [2]. Accumulated evidence suggests that LRRK2 may play a key role in axonal extension, autophagy, proliferation, and survival of neurons through its kinase activity [6-9]. In spite of the proposed mechanisms for neurodegeneration in vitro, the mechanism by which LRRK2 mutations affect DA neurons in patients and model animals in vivo is still far from conclusive. As a mammalian PD model, transgenic (TG) mice expressing the R1441G mutation at the LRRK2 ROC domain reportedly show reduction of locomotive ability and diminished dopamine release [10]. The R1441C knock-in (KI) mouse, on the other hand, appears normal in steadystate, although a reduction of amphetamine-induced locomotor activity and impaired D2 receptor function have been observed [11]. Four different TG mouse lines expressing the G2019S mutation in the LRRK2 kinase domain, have been reported [12-15]. Two of them displayed increased ambulatory activity but the others did not. In terms of pathology, only one of them showed degeneration of DA neurons accompanied by abnormal autophagy, whereas two others showed increased tau-phosphorylation or promotion of tubulin polymerization associated with Golgi fragmentation. Temporal overexpression of G2019S LRRK2 in rat reportedly impairs dopamine reuptake, leading to enhanced locomotive activity [16]. In contrast to the extensive analysis of R1441G, R1441C, and G2019S TG mice, no LRRK2 TG rodent model with the I2020T mutation has ever been reported. G2019S and I2020T, despite being mutations affecting neighboring residues, have been known to have distinctive effects on the LRRK2 molecule, as reflected in kinase activity and susceptibility to post-translational degradation [17,18]. In Drosophila, TG flies expressing the I2020T LRRK2, or its homologue I1915T LRRK, have been reported to show either DA neuron loss leading to unusual locomotive activity or a decrease of neuromuscular junction boutons [19-21]. In the present study, we investigated a TG mouse strain expressing I2020T LRRK2 in DA neurons. The TG mouse exhibits impaired locomotive ability, a reduced striatum dopamine content, fragmented Golgi apparatus, and an elevated degree of tubulin polymerization. Furthermore, the tyrosine hydroxylase-positive (TH+) primary neurons of the TG mouse show increased vulnerability and shortened neurites. Results Generation of I2020T LRRK2 TG mice We obtained 9 independent mouse lines harboring the V5-tagged human LRRK2 cDNA with the I2020T mutation and single nucleotide polymorphisms (SNPs) of the Sagamihara family patients (Figure 1A). One of them (line 41) was found by genomic Southern analysis to harbor about 5 copies of the transgene at two insertion sites, one of which had multiple tandem insertions (Additional file 1. figures S1A and S1B). Although some other lines harbored more copy numbers than line 41, the latter line was the only line stably expressing the full-length I2020T LRRK2 in brain, and was therefore used throughout in this study. The line 41 TG mice appear healthy from birth and develop with normal weight and fertility, and live births show the expected Mendelian ratio. The TG mice expressed I2020T LRRK2 mRNA in all of the tissues analyzed (Figure 1B). Western analysis of whole brain using an antiV5 antibody detected the I2020T LRRK2 full-length proteins (Figure 1C). Immunofluorescence staining of brain tissues using anti-V5, anti-TH, and anti--III tubulin antibodies indicated that the neuronal cells including TH+ neurons of the substantia nigra compacta (SNc), ventral tegmental area, and olfactory bulb expressed the I2020T LRRK2 proteins (Figure 1D). Quantitative PCR revealed that the level of expression of LRRK2 mRNA in the line 41 TG mice was about 1.5-, 1.4-, and 1.2-fold that of NTG control mice in the whole brain, striatum, and midbrain, respectively (Additional file 1. figure S2). Measurement of the immunofluorescence intensity of individual TH+-neurons in the substantia nigra with MJFF2 recognizing both human LRRK2 and mouse LRRK2 revealed that the level of LRRK2 protein expression in TH+-neurons of TG mice was about 1.3-fold that of TH+-neurons in NTG control mice (Additional file 1. figure S3). I2020T LRRK2 TG mice exhibit impaired locomotive ability To assess the locomotive ability of I2020T LRRK2 TG mice, we subjected them to the beam test. While TG mice aged 23 weeks traversed the narrow beam, they exhibited slips more frequently (in terms of both per time and per step) than their non-TG (NTG) littermates (Figure 2A). This impaired locomotive ability was also observed in 43-week-old TG mice (slips per time), but was undetectable in 73-week-old TG mice. In the rotarod test, I2020T TG mice (...truncated)